scSLAM-seq
scSLAM-seq was developed by [Erhard2019] and is the single-cell adaptation of thiol(SH)-linked alkylation for metabolic sequencing of RNA (SLAM-seq) [Herzog2017]. Similar to NASC-seq, scSLAM-seq is based on the Smart-seq protocol [Picelli2013]. Smart-seq experiments generate single or pairs of FASTQs for each cell sequenced, which dynast processes simultaneously.
Sequencing technology: Smart-Seq2
Induced conversion: T>C
Alignment
Here, we assume the appropriate STAR index has already been built (see Building the STAR index with ref). Since we have multiple sets of FASTQs, we need to prepare a FASTQ manifest CSV, instead of providing these as an argument to dynast align
. The manifest CSV contains three columns where the first column is a unique cell name/ID, the second column is the path to the first FASTQ, and the third is the path to the second FASTQ. For single-end reads, the third column can be a single -
character. Here is an example with two cells:
cell_1,path/to/R1.fastq.gz,path/to/R2.fastq.gz
cell_2,path/to/R1.fastq.gz,-
Then, we use this manifest as the input to dynast align
.
dynast align -i path/to/STAR/index -o path/to/align/output -x smartseq --strand unstranded manifest.csv
Note that we provide --strand unstranded
because the Smart-seq protocol used with scSLAM-seq produces unstranded reads. This will run STAR alignment and output files to path/to/align/output
.
Quantification
The alignment BAM is generated at path/to/align/output/Aligned.sortedByCoord.out.bam
, which we provde as input to dynast count
. We also need to provide the gene annotation GTF that was used to generate the STAR index to -g
.
dynast count -g path/to/GTF.gtf --barcode-tag RG path/to/align/output/Aligned.sortedByCoord.out.bam -o path/to/count/output --conversion TC --strand unstranded
Note that we provide --strand unstranded
again because the Smart-seq protocol used with scSLAM-seq produces unstranded reads. This will quantify all RNA species and write the count matrices to path/to/count/output/adata.h5ad
.